Tipos de aparcabicis y criterios de utilización

2.2.1 Plasmid DNA extraction

4mL Lysogeny broth (LB) medium (Table 2.3) with relevant antibiotics (Table 2.4) was inoculated with a single colony and was incubated overnight at 37°C with shaking. The cell culture was then pelleted by centrifugation at 5,000rpm for 2min and was re-suspended in a 300µl solution I buffer (10mM Tris-HCl, 1mM EDTA pH 8.0) to lyse cells. Subsequently, 300µl freshly prepared solution II (0.2M NaOH, 1% SDS) was added into the cell lysis and was mixed by inverting. Followed by adding 300µl ice-cold solution III (3M potassium acetate, 5M acetic acid), precipitated proteins were pelleted by centrifugation at 4°C 12,000rpm for 10min. Subsequently, the supernatant was transferred to a new Eppendorf tube and was mixed with the same volume of isopropanol. The plasmid was then pelleted by centrifugation at 4°C 12,000 rpm for 10min. After washed with 500µl 75% ethanol for two times, the pellet containing plasmid DNA was re-suspended in 30µl ddH2O and stored at -20°C.

Table 2.3: LB medium Compositions Concentrition

Bacto Tryptone 10g/l

Yeast extract 5g/l

Adjust pH to 5.7 with KOH. For sodium, 1.5g/l agar was added

Table 2.4: List of antibiotics used for selection

Name Stock concentration Work concentration Solvent

Ampicilin 100mg·ml-1 _100μg·ml-1 _ddH2O

Gentamycin 15mg·ml-1 _15μg·ml-1 _ddH2O

Kanamycin 50mg·ml-1 _50μg·ml-1 _ddH2O

Rifampilin 25mg·ml-1 _100μg·ml-1 _DMSO

2.2.2 DNA fragments manipulation

The plasmid DNA or DNA fragments were digested by restriction enzymes produced by NEB Biolabs or Fermentas according to the instruction from the manufacturers. The products of digestion were separated by gel electrophoresis running in the TAE buffer (Table 2.5), and then the target DNA fragments were recycled by using a gel

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recycle kit (GeneJET Gel Extraction Kit, Thermo Fischer Scientific). Subsequently, DNA fragments were jointed together by T4 DNA ligase produced by NEB Biolabs.

Table 2.5: TAE buffer

Compositions Concentrition Tris-Acetate 40mM

EDTA 1mM

Adjust pH to 8.0 with HCl

2.2.3 Chemically competent Escherichia coli cells preparation and transformation 4ml LB medium was inoculated with a single colony and incubated overnight at 37°C with shaking. 1ml cell culture was subsequently inoculated into a 500ml LB medium with 5ml sterilized 1M MgCl2 solution. The culture was then incubated at 28°C, with shaking, for around 4h until OD600 reached 0.5~0.7. The cell culture was then cooled down on the ice for 10min. The cells were then harvested by centrifugation at 4,500rpm for 10min at 4°C and were then dissolved in the 80ml pre-cooled TB buffer (10mM Pipes, 15mM CaCl2, 250mM KCl, pH6.7 with KOH and 55mM MnCl2). Finally, aliquots of 50µl competent cells were frozen in liquid nitrogen and stored at -80°C. For transformation, around 100ng DNA was added into 50µl aliquot of competent cells. The aliquot was then incubated on ice for 30min. After a heat-shock treatment at 42°C for 90s, the aliquot was quickly cooled down on ice for 5min, and 500µl LB medium was added. Subsequently, the cells were incubated at 37°C for 45min and were plated on selection media and were grown overnight at 37°C.

2.2.4 DNA extraction from plants

Two methods were used to extract genomic DNA of Arabidopsis according to the requirements on the DNA quality in different experiments. A protocol was used to isolate a small amount of unpurified DNA. 4-10mm2 _{leaf disk was harvested and} homogenized by a pestle in 100µl rough DNA extraction buffer (Table 2.6). After centrifugation at 14,000rpm for 4min, 80µl supernatant was transferred to a new tube and was mixed with 80µl isopropanol. DNA was pelleted by centrifugation at 13.000rpm for 5min. The pellet was washed by 500µl 75% ethanol for two times and was finally dissolved in 20μl ddH2O. DNA samples were stored at -20 °C.

Table 2.6: Rough DNA extraction buffer

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Tris 200mM

NaCl 150mM

EDTA 25mM

SDS 0.5%

Another method was used to isolate a large amount of purified and clean DNA. Around 20mg leaf material was harvested and frozen in liquid nitrogen. Samples were homogenized at 25Hz for 1.5min by a Retsch cyro grinding mill (MM301, Retsch). 500µl clean DNA extraction buffer (Table 2.7) and 66µl 10% SDS were added into the samples and incubated at room temperature (RT) for 10min. After centrifugation at 13,000rpm for 15min, the supernatant was transferred into a new tube and mixed with 166µl KOAc solution (Table 2.8). The same volume of isopropanol was then added into the mixture and was incubated on ice for 20min. After centrifugation at 16,000rpm for 20min, the supernatant was removed, and the pellet was washed with 500µl 70% ethanol for two times. Finally, pellets were dissolved in 100µl TE buffer containing 1µg/ µl RNase A.

Table 2.7: Clean DNA extraction buffer

Compositions Concentrition

Tris 0.1M

NaCl 0.5M

EDTA 0.05M

Polyvinylpyrrolidone PVP-40 1%

Table 2.8: KOAc solution

Compositions Concentrition

Potassium acetate 3M

acetic acid 2M

2.2.5 Polymerase chain reaction and DNA sequencing

Primers for polymerase chain reaction (PCR) were designed by the software, PrimePrimer. The components and thermos-cycling conditions for standard PCR reaction were listed in Table 2.9 and Table 2.10. The annealing temperatures of primers were designed around 58°C. Sequences of PCR products were verified by the

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second generation DNA sequencing technology by LGC Genomics (Berlin, Germany). Samples were provided according to the requirements of the company.

Table 2.9: Standard PCR reaction

Compositions Volume DNA polymerase (Taq or

Phusion) 0.2μl 10x Buffer 2μl 10mM dNTPs 0.5μl 10μM forward primer 1μl 10μM reverse primer 1μl DNA template 1μl ddH2O 14.3μl

Table 2.10: Program for the standard PCR reaction

Steps Temperature (°C) Time (s)

Pre-denaturing 95 120

Denaturing 95 15

Annealing 58 15

Extending 72; go back to denaturing 29

cycle 60 per 1kb

Additional

extending 72 120

2.2.6 The overlap extension PCR

For the linkage of two or three PCR fragments, the overlap extension PCR was applied, which generally includes three round PCR reactions. The first-round PCR was performed as the standard PCR reaction as described in 2.2.5 to amplify the single fragments from Arabidopsis genomic DNA or from cDNA. PCR products were then purified by a gel recycle kit after electrophoresis. The fragments were jointed together to form the full-length fusion product in the second-round PCR. No primers were added in this round, the linkage of the single fragments took place, relying on the complementary sequences within the fragments. The reaction mixtures

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contained PCR products from the first round, 5xHF-buffer, dNTP mix (10mM each) and Phusion® (2U/μl). The thermos-cycling conditions for the second round were similar to the standard PCR, except that the annealing temperature was reduced to 50~55°C and the cycle number was reduced to 10. The last reaction was a standard PCR reaction to amplify the full length of target DNA by using the PCR products of the second round as the template. The PCR products from the last-round PCR was then separated by electrophoresis and recycled by a DNA gel recycle kit.